While adoptive immunotherapy is a promising treatment for a variety of cancers, progress has been impeded by the lack of a reproducible and economical method for generating therapeutic numbers of antigen-specific CTL. To address this issue, we developed an HLA-Ig, bead-based artificial Antigen-Presenting Cell (aAPC) which reliably induced and expanded melanoma and other tumor antigen-specific CTL in vitro. During the last funding period, we developed the aAPC into a multifunctional platform technology. We demonstrated that aAPC-induced melanoma antigen, Mart-1 specific CTL functioned in both in vivo protection and treatment models and that MHC-Ig based aAPC could also be used for in vivo administration to stimulate low affinity melanoma- specific CTL. In addition we characterized CTL polyfunctionality in different human antigen-specific CTL populations stimulated with DC or APC. We also developed a novel assay that uses the fluorescence of quantum dots to characterize TCR microclusters on cells. The goals of the proposed research are: to optimize aAPC-induced CTL function in vivo by studying the characteristics of CTL populations that track to and eliminate tumors, to understand mechanistically how in vivo aAPC administration increases CTL function and to understand the regulation of CTL polyfunctionality. These studies will facilitate our understanding of how to optimize CTL for adoptive immunotherapy and further develop the potential uses of HLA-Ig-based aAPC for melanoma immunotherapy.